Process for the preparation of poly-alpha-glutamic acid and derivatives thereof

ABSTRACT

The invention relates to an improved process for the preparation of poly-α-glutamic acids which comprises the polymerization of tertiary γ-esters of α-glutamic acid N-carboxy anhydride with appropriate solvents and initiators, followed by acid hydrolysis of the resulting poly-α-glutamic acid-γ-ester. The process is particularly advantageous in that it allows one to carefully control the molecular weight of the resulting poly-α-glutamic acid. The invention also relates to poly-α-glutamic acids capped at the amino terminus with carboxylic acids or amino acids and to a process for the preparation thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Patent Application No. 60/813,787 filed Jun. 15, 2006 andpriority to European Patent Application No. 06012351.0 filed Jun. 15,2006; which applications are incorporated by reference herein in theirentirety.

BACKGROUND

1. Technical Field

The present invention relates to a process for the preparation ofpoly-α-glutamic acid and derivatives thereof, and compounds therefrom.

2. Description of the Related Art

Poly-α-glutamic acid of formula (I)

is currently used as drug delivery system, for example for thepreparation of conjugates with a wide variety of drugs containing aprimary or secondary amino group as well as primary, secondary ortertiary hydroxy groups. The conjugation can occur directly or through asuitable linker, for example an amino acid or a hydroxyacid. See, forexample, EP0932399, Advanced Drug Delivery Reviews, Volume 54, Issue 5,2002, Pages 695-713 and Journal of Controlled Release, Volume 74, Issues1-3, 2001, Pages 243-247.

Poly-α-glutamic acid can be synthesized by polymerization of suitablyprotected glutamic acid. U.S. Pat. No. 3,635,909 discloses, inter alia,the polymerization of D-glutamic acid-γ-(tert-butyl)ester N-carboxyanhydride in a 1,2-dichloroethane/1,4-dioxane mixture using sodium4-methyl-2-pyrrolidone as initiator.

BRIEF SUMMARY

Briefly stated, compounds and processes for the preparation ofpoly-α-glutamic acid and derivatives thereof are provided.

In an embodiment, the present invention provides a process for thepreparation of poly-α-glutamic acid of formula (I)

wherein the symbol * indicates a chiral center and n is between 60 and310, so that the poly-α-glutamic acid has a molecular weight rangingfrom 8,000 to 40,000 Da

the process comprising the steps of:

a) polymerization of a tertiary γ-ester of α-glutamic acid N-carboxyanhydride of formula (II)

wherein the symbol * is as defined above and R is selected from t-butyl,1,1-dimethylpropyl and 1,1-dimethylbutyl

in water or in an organic solvent selected from: tetrahydrofuran,1,4-dioxane, dimethylformamide, 1,4-dioxane/DMF and1,4-dioxane/tetrahydrofuran mixture with an initiator selected frompotassium tert-butoxide, sodium methoxide, diisopropylethylamine,1,8-diazabicyclo[5,4,0]undec-7-ene, dimethylaminopyridine and L-glutamicacid-γ-tert-butylester, to give a compound of formula (III)

wherein * and R are as defined above and R′ is hydrogen when theinitiator is selected from diisopropylethylamine,1,8-diazabicyclo[5,4,0]undec-7-ene, 4-dimethylaminopyridine, glutamicacid dimethyl ester and glutamic acid-γ-tert-butyl ester or R′ is at-butyl or methyl group when the initiator is potassium tert-butoxideand sodium methoxide respectively; and followed by

b) acid hydrolysis of the γ- and α-ester groups to give a compound offormula (I).

In an embodiment, the present invention provides a process for thepreparation of a poly-α-glutamic acid derivative of formula (IV)

wherein the symbol * indicates a chiral center and n is an integercomprised between 60 and 310 and R₁CO— is selected from:

(C₁-C₁₀)alkylcarbonyl;

(C₄-C₈)cycloalkylcarbonyl;

(C₂-C₆)carboxyalkylcarbonyl;

(C₆-C₁₀)arylcarbonyl;

(C₆-C₁₀)aryl(C₁-C₁₀)alkylcarbonyl;

(C₁-C₁₀)alkyl(C₆-C₁₀)arylcarbonyl;

(C₅-C₁₀)heteroarylcarbonyl and (C₅-C₁₀)heteroaryl(C₁-C₁₀)alkylcarbonylwherein the heteroaromatic ring contains one or more nitrogen, oxygen orsulphur atoms; and

D- or L-amino acid and non-natural amino acid residues; the processcomprising the steps of:

a) polymerization of a tertiary γ-ester of an α-glutamic acid N-carboxyanhydride of formula (II)

wherein * is as defined above and R is selected from t-butyl,1,1-dimethylpropyl and 1,1-dimethylbutyl

in water or in an organic solvent selected from: tetrahydrofuran,1,4-dioxane, dimethylformamide, 1,4-dioxane/DMF and1,4-dioxane/tetrahydrofuran mixtures with an initiator selected frompotassium tert-butoxide, sodium methoxide, diisopropylethylamine,1,8-diazabicyclo[5,4,0]undec-7-ene, dimethylaminopyridine and L-glutamicacid-γ-tert-butylester, to give a compound of formula (III);

b) reaction of a compound of formula (III)

obtained according to step a) above with a carboxylic acid R₁COOH, or anacyl chloride R¹COCl or an anhydride (R₁CO)₂O wherein R₁ is as definedabove, in the presence of a dehydrating agent to give a compound offormula (V)

and

c) the hydrolysis of the compound of formula (V) to give a compound offormula (IV).

In an embodiment, the present invention provides a poly-α-glutamic acidderivative of formula (IV)

wherein the symbol * indicates a chiral center and n is an integercomprised between 60 and 310 and R₁CO— is selected from:

(C₁-C₁₀)alkylcarbonyl;

(C₄-C₈)cycloalkylcarbonyl;

(C₆-C₁₀)arylcarbonyl;

(C₆-C₁₀)aryl(C₁-C₁₀)alkylcarbonyl;

(C₁-C₁₀)alkyl(C₆-C₁₀)arylcarbonyl;

(C₅-C₁₀)heteroarylcarbonyl and (C₅-C₁₀)heteroaryl(C₁-C₁₀)alkylcarbonylwherein the heteroaromatic ring contains one or more nitrogen, oxygen orsulphur atoms; and

D- or L-amino acid and non-natural amino acid residues as well as theirsalts with inorganic acids or bases.

In an embodiment, the present invention provides a poly-α-glutamic acidderivative of formula (V)

wherein the symbol * indicates a chiral center and n is an integercomprised between 60 and 310, R is selected from t-butyl,1,1-dimethylpropyl and 1,1-dimethylbutyl, R′ is hydrogen or a t-butyl ormethyl group and R₁CO is selected from:

(C₁-C₁₀)alkylcarbonyl;

(C₄-C₈)cycloalkylcarbonyl;

(C₆-C₁₀)arylcarbonyl;

(C₆-C₁₀)aryl(C₁-C₁₀)alkylcarbonyl;

(C₁-C₁₀)alkyl(C₆-C₁₀)arylcarbonyl;

(C₅-C₁₀)heteroarylcarbonyl and (C₅-C₁₀)heteroaryl(C₁-C₁₀)alkylcarbonylwherein the heteroaromatic ring contains one or more nitrogen, oxygen orsulphur atoms; and

D- or L-amino acid and non-natural amino acid residues.

These and other aspects of the present invention will become apparentupon reference to the following detailed description.

DETAILED DESCRIPTION

The present disclosure shows that appropriate choice of the solvent andof the initiator allows the control of the molecular weight ofpoly-α-glutamic acid prepared from tertiary γ-esters of α-glutamic acidN-carboxy anhydride. Accordingly, in one aspect the invention relates toa process for the preparation of poly-α-glutamic acid of formula (I)

wherein n is an integer comprised between 60 and 310, so that thepoly-α-glutamic acid has a molecular weight ranging from 8,000 to 40,000Da, preferably from 10,000 to 35,000 Da, more preferably from 13,000 to16,000 Da, and with a polydispersity index usually ≦2, preferably ≦1.5,

said process comprising the steps of:

a. polymerization of a tertiary γ-ester of α-glutamic acid N-carboxyanhydride of formula (II)

wherein R is selected from t-butyl, 1,1-dimethylpropyl- and1,1-dimethylbutyl- and is preferably a t-butyl group either in water orin an organic solvent selected from tetrahydrofuran (THF), 1,4-dioxane,dimethylformamide (DMF), 1,4-dioxane/DMF and 1,4-dioxane/tetrahydrofuranmixtures with an initiator selected from potassium tert-butoxide(t-BuOK), sodium methoxide (MeONa), diisopropylethylamine,1,8-diazabicyclo[5,4,0]undec-7-ene (DBU), 4-dimethylaminopyridine(DMAP), glutamic acid dimethyl ester, and glutamic acid-γ-tert-butylester, to give a compound of formula (III)

wherein n and R are as defined above and R′ is hydrogen when theinitiator is selected from diisopropylethylamine,1,8-diazabicyclo[5,4,0]undec-7-ene (DBU), 4-dimethylaminopyridine(DMAP), glutamic acid dimethyl ester and glutamic acid-γ-tert-butylester or R′ is a t-butyl or methyl group when the initiator is t-BuOKand MeONa respectively; and followed by

b. acid hydrolysis of the γ- and α-ester groups to give a compound offormula (I).

Throughout the specification, the symbol * indicates a chiral center andthe term glutamic acid comprises both the L- and D-form, either as pureisomers or as racemic mixture.

Step a) is usually carried out at a temperature ranging from 10 to 50°C., with a concentration of the γ-ester of α-glutamic acid N-carboxyanhydride ranging from 0.1 to 0.3 M. According to a preferredembodiment, the process is carried out using 1,4-dioxane as the solventand DBU as the initiator.

The molar ratio of γ-ester of α-glutamic acid N-carboxy anhydride toinitiator usually ranges from 2 to 25; the molar ratio of 7-ester ofα-glutamic acid N-carboxy anhydride to α-glutamic acid γ-ester is 100.

Step b) can be carried out using any reagent conventionally used for theremoval of tert-butyl esters, as exemplified by those described in“Protective groups in organic synthesis” Third edition, Theodora W.Greene and Peter G. M. Wuts, a Wiley-Interscience publication—John Wiley& Sons, Inc, page 406-408 and references therein. Preferred conditionsinclude trifluoroacetic acid (TFA), formic acid and water/formic acidmixtures at a temperature ranging from 20 to 60° C.; TFA is used inamount of 100 v/w, while formic acid and water/formic acid mixtures areused in amounts ranging from 20 to 50 v/w.

The process of the invention is suitable for preparing both the D and Lform of poly-α-glutamic acids, in particular the L form. Moreover, thepoly-α-glutamic acids prepared according to the invention can be easilyconverted into their acidic or basic salts by reaction with inorganicacids, such as hydrochloric acid, hydrobromic acid, sulphuric acid,nitric acid, perchloric acid or with inorganic bases such as alkaline oralkaline-earth metal hydroxides and ammonium hydroxide.

The process of the invention provides poly-α-glutamic acid with a weightaverage molecular weight (M_(w)) determined by Gel PermeationChromatography combined with Multi Angle Laser Light Scatteringdetection, ranging within the values defined above, preferably between13,000 and 16,000 Da and with a polydispersity index ≦1.5. This isparticularly advantageous, since the procedures disclosed in theliterature, through different γ-protecting groups (such as benzyl,methyl or ethyl; see Polymer monographs, Volume 9: H. Block,Poly(γ-benzyl-L-glutamate) and other glutamic acid containing polymers.Edited by B. Huglin, University of Salford), afford materials ofuncontrolled and very high molecular weight, which often requirechromatographic separation to isolate poly-α-glutamic acids having adesired molecular weight range.

Moreover, the process of the invention allows avoidance of spontaneousformation of a pyroglutamic ester at the amino terminus, which usuallyoccurs during polymerization using, for example, γ-benzyl, γ-methyl orγ-ethyl glutamic acid ester N-carboxy anhydride. In fact, currentlyavailable poly-α-glutamic acids exhibit either a pyroglutamic terminus,or an amino terminus or a mixture thereof. Suppression of formation of apyroglutamic terminus is achieved in particular when a compound offormula (II) wherein R is a t-butyl group is used as the startingmaterial. Poly-α-glutamic acids γ-esters with a free amino terminus canthus be reacted either with stoichiometric amounts of pyroglutamic acidor with other suitable acids, so as to achieve 100% conversion into thedesired N-capped poly-α-glutamic acid.

Accordingly, in another aspect the present invention provides also aprocess for the preparation of poly-α-glutamic acid derivatives offormula (IV)

wherein n is as defined above and R₁CO— is selected from:

(C₁-C₁₀)alkylcarbonyl; (C₄-C₈)cycloalkylcarbonyl;(C₂-C₆)carboxyalkylcarbonyl; (C₆-C₁₀)arylcarbonyl;(C₆-C₁₀)aryl(C₁-C₁₀)alkylcarbonyl; (C₁-C₁₀)alkyl(C₆-C₁₀)arylcarbonyl;(C₅-C₁₀)heteroarylcarbonyl and (C₅-C₁₀)heteroaryl(C₁-C₁₀)alkylcarbonylwherein the heteroaromatic ring contains one or more nitrogen, oxygen orsulphur atoms; D- or L-natural and non-natural amino acid residues

said process comprising reacting a compound of formula (III)

obtained according to step a) above with a carboxylic acid R₁COOH in thepresence of a dehydrating agent, or with an activated carboxylic acid,such as an acyl chloride R₁COCl or an anhydride (R₁CO)₂O, wherein R₁CO—is as defined above, to give a compound of formula (V)

and hydrolyzing the compound of formula (V) to a compound of formula(IV).

In the compounds of formula (IV) and (V):

examples of (C₁-C₁₀)alkylcarbonyl are acetyl and butyryl;

examples of (C₄-C₈)cycloalkylcarbonyl are cyclopropylcarbonyl,cyclobutanecarbonyl, cyclohexylcarbonyl;

example of (C₂-C₆)carboxyalkylcarbonyl is succinyl;

examples of (C₆-C₁₀)aryl carbonyl are benzoyl, 1-naphthoyl, 2-naphthoyl;

examples of (C₆-C₁₀)aryl(C₁-C₁₀)alkylcarbonyl are phenylacetyl andphenylbutyryl;

examples of (C₁-C₁₀)alkyl(C₆-C₁₀)arylcarbonyl are o-, m- and p-tolyl;

examples of (C₅-C₁₀)heteroarylcarbonyl are nicotinoyl,N-methylpyrrole-3-carbonyl, 3-thiophenecarbonyl and 3-quinolinecarbonyl;

example of (C₅-C₁₀)heteroaryl(C₁-C₁₀)alkylcarbonyl is 3-pyridyl acetyl;

examples of D- or L-natural amino acid residues are those deriving fromglycine, alanine, valine, leucine, isoleucine, serine, threonine,lysine, pyroglutamic acid, phenylalanine, tryptophan, cysteine;

examples of non-natural amino acid residues are those deriving fromβ-alanine, α,α-dimethylglycine, α-phenylglycine, homophenylalanine,3-amino-3-(4-methylphenyl)propionic acid, and2-(1-aminocyclopentyl)acetic acid.

The reaction between compound (III) and the acid R₁COOH is carried outin the presence of a dehydrating agent commonly used in peptidechemistry, like carbodiimides, such as dicyclohexylcarbodiimide anddiisopropylcarbodiimide, uronium salts, such as2-1H-7-azabenzotriazol-1-yl-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (HATU),2-(1H-benzotriazol-1-yl)-1,1,3,3-N,N,N′,N′-tetramethyluronium-hexafluorophosphate(HBTU) and 2-(1H-6-chlorobenzotriazol-1-yl)-1,1,3,3-N,N,N′,N′-tetramethyluronium-hexafluorophosphate (HCTU), phosphonium salts,such as benzotriazol-1-yloxytris(pyrrolidino)phosphoniumhexafluorophosphate (PyBOP), and the like. When a natural or non-naturalamino acid is used as the capping agent, its amino group is preferablyprotected by a suitable protecting group, preferably thet-butoxycarbonyl protecting group. Subsequent acidic hydrolysis of theN-capped poly-α-glutamic acid-γ-ester as described in step b) above thenprovides the N-capped poly-α-glutamic acid. If desired, the compounds offormula (IV) can be converted into their acidic or basic salts byreaction with inorganic acids, such as hydrochloric acid, hydrobromicacid, sulphuric acid, nitric acid, perchloric acid or with inorganicbases such as alkaline or alkaline-earth metal hydroxides and ammoniumhydroxide.

The compounds of formula (IV) and (V) are disclosed as another aspect ofthe present invention.

A particularly preferred embodiment is a compound of formula (IV)wherein R₁CO is a D- or L-pyroglutamic acid residue and the content ofthe N-terminus free amine is lower than 1% w/w, preferably lower than0.2% w/w.

The process described above for the preparation of a compound of formula(IV) has general applicability, that is to say that, further to acarboxylic acid R₁COOH, or an activated carboxylic acid, such as an acylchloride R₁COCl or an anhydride (RICO)₂O, any compound able to reactwith the —NH₂ terminus can be used for the reaction with the compound offormula (III). Examples of such compounds are aldehydes, ketones,α,β-unsaturated ketones, sulfonyl chlorides and nitriles, which can bereacted with compound (III) according to methods known to the skilledchemist. According to a further embodiment of the invention either thecarboxylic acid, or activated carboxylic acid, or anhydride, or compoundable to react with the —NH₂ terminus may also bear at least a group,such as a hydroxy, an amino or a thio group, which can be furtherfunctionalized with a moiety suitable for modulating the pharmacokineticproperties of poly-α-glutamic acid. Therefore, another aspect of thepresent invention is also a poly-α-glutamic acid substituted at the —NH₂terminus with a group which is able to be further functionalized with amoiety suitable for modulating the pharmacokinetic properties ofpoly-α-glutamic acid.

Embodiments of the invention will be hereinafter illustrated in greaterdetail in the following examples. The examples are offered by way ofillustration and not by way of limitation.

EXAMPLES Example 1 Step a Preparation of L-α-Glu-NCA-γ-tBu ester

40.17 g of triphosgene (0.135 mol; phosgene/L-Glu-γ-tBu ester 2.74/1)and 862.5 mL of THF were placed under nitrogen into a 1 L jacketedreactor, equipped with mechanical stirrer. The reactor was thermostatedat 20° C. and the mixture was stirred until complete dissolution oftriphosgene (a few minutes are necessary), thereafter 30 g ofL-α-Glu-γ-tBu ester (0.148 mol) were added in a single portion and quiterapidly. The resulting suspension was allowed to react for 2 hours at20° C. At the beginning exothermicity was observed (≈4° C.), then thesolid dissolved as the reaction proceeded: after about 30 min a clearsolution was obtained.

At the end of the reaction the solvent was evaporated under reducedpressure, keeping the jacket temperature not higher than 20° C.: thedistillation was quite tumultuous. The distillation was continued untila dense and oily residue was obtained, without solids (about 80 mL ofresidue expected). At the end of the distillation 750 mL of n-heptanewere dropped in over about 15 min and the mixture was stirred undernitrogen at 20/23° C. for 1 hour until complete crystallization of theproduct. Usually, crystallization begins after addition of 150 mL ofn-heptane.

The resulting pure white solid was filtered on a buchner funnel andwashed with 3×90 mL of n-heptane, then dried in the oven under dynamicvacuum at 20/25° C. for no longer than 20 hours. The solid must be usedas soon as possible and must be stored under static vacuum in thepresence of silica gel.

Starting from 30 g of L-α-Glu-γ-tBu ester, 23.3 g of L-α-Glu-NCA-γ-tBuester were obtained (68.8% yield).

Step b Polymerization of α-L-glutamic acid-γ-(tert-butyl)ester N-carboxyanhydride) (NCA) to poly-α-L-glutamic acid-γ-(tert-butyl)ester

1,8-Diazabiciclo[5.4.0]undec-7-ene free base (DBU; MW 152.24; 585 mg,3.84 mmol) was dissolved in 1,4-dioxane (5 mL from the total amount) infew minutes.

In a 1 L jacketed glass reactor 1,4-dioxane (550 mL) and α-L-glutamicacid-γ-(tert-butyl)ester N-carboxy anhydride (NCA) (22 g, 96 mmol;NCA/DBU=25) were mixed at 25° C. until dissolution was complete.

The resulting solution was added very quickly with the previouslyprepared DBU solution. A precipitate instantly appeared and lowexothermicity was observed (about 3° C.).

The mixture was stirred for 4 hours, then water (1100 mL) was slowlypoured in over 30 to 40 minutes. After a further 30 minutes stirring at25° C. the precipitated solid was collected by filtration and washedwith water (3×50 mL). The recovered white solid was then dried undervacuum at 40° C. to yield 16.4 g of pure material, t-BuPG (92% recovery;DP_(n)=137.2, number average degree of polymerization; M_(n)=25,400 Da,number average molecular weight from ¹H-NMR).

Step c Hydrolysis of poly-α-L-glutamic acid-γ-(tert-butyl)ester topoly-α-L-glutamic acid

Solid poly-α-L-glutamic acid-γ-(tert-butyl)ester (4 g) was suspended informic acid 99% (80 mL, 20 volumes) under nitrogen atmosphere in a 100mL jacketed glass reactor. The mixture was stirred at 60° C. for 5hours. After about a 30-minute heating the suspension turned intosolution, then a solid began to precipitate.

Most of the formic acid was then removed by distillation under reducedpressure (P=15 torr; internal temperature always below 60° C.). Theresidual formic acid was evaporated off by azeotropic distillation withtoluene (2×40 mL; T<40° C.).

The resulting residue was suspended in water (40 mL) and the mixture wascooled to 3-5° C., then 30% w/w NaOH was carefully added to reach pH 8.Once the pH was stable at 23-25° C., the resulting solution was filteredthrough a 0.22 μm microfilter.

The clear solution was acidified by means of sulphuric acid up to pH2.5, then the resulting suspension was stirred for 2 hours. Theprecipitated solid was filtered off and dried under vacuum (30° C.overnight). A white cake was recovered (2.7 g) with a M_(w)=13,900 Daand polydispersity 1.04 (GPC-MALLS).

Example 2

α-L-glutamic acid-γ-(tert-butyl)ester N-carboxy anhydride (3.95 g, 17.2mmol) was dissolved, under nitrogen stream, in THF (100 mL) to reach aconcentration of 0.039 g/mL (0.17 M). The stirring speed was set to 800rpm.

Then DBU (105 mg, A/I=25) was added by a syringe very quickly and themixture was stirred for 4 hours at 22-23° C. A slight exotherm wasobserved soon after addition of the initiator.

Afterwards, the reaction mixture was concentrated to dryness and theresidue was rinsed with fresh 1,4-dioxane (100 mL). Two volumes of waterwere then added to the reaction mixture which was stirred for 30minutes. The precipitated solid was collected, washed with water anddried under vacuum at 40° C. for at least 12 hours.

2.85 g of poly-α-L-glutamic acid-γ-(tert-butyl)ester (90% recovery) as awhite powder with M_(n)=34,100 Da and DP_(n)=184 (by ¹H-NMR) wereobtained.

The resulting poly-α-L-glutamic acid-γ-(tert-butyl)ester was thenhydrolyzed in hot 99% HCOOH according to the procedure described in stepc of Example 1, furnishing about 1.7 g of poly-α-L-glutamic acid (84%recovery) with M_(w)=25,700 and polydispersity=1.2 according to GelPermeation Chromathography coupled with Multi Angle Laser LightScattering analysis.

Example 3 General Procedure for the Polymer Capping Reaction

A typical poly-α-L-glutamic acid-γ-(tert-butyl)ester, obtained accordingto an experimental procedure herein described (see Example 1 and 2),contains about 1% moles of free amino groups at the N-terminus, asdetermined by ¹H-NMR analysis. This percentage was used to calculate themolar equivalents of the suitable acid to add to poly-α-L-glutamicacid-γ-(tert-butyl)ester for its capping reaction.

Poly-α-L-glutamic acid-γ-(tert-butyl)ester was dissolved in THF (20mL/g) then a catalytic amount of 4-dimethylaminopyridine (DMAP; about5-10% moles with respect to the added acid) was added. The suitable acid(10 molar equivalents) was added to the previous mixture anddiisopropylcarbodiimide (DIPC, 50% molar excess with respect to the acidadded) was dropwise added in about 10 min. The solution was stirred for2 hours.

Two volumes of water were then added and the resulting mixture wasstirred for 30 min. The precipitated solid consisting ofN-acyl-poly-α-L-glutamic acid-γ-(tert-butyl)ester was collected and thensuspended in formic acid (20 volumes). After stirring at 60° C. for 2 h,two volumes of water were added and the resulting mixture was stirredfor 30 min. The precipitated solid was removed by filtration, washedwith water and dried under vacuum (30° C., overnight) to yield asuitably N-capped poly-α-L-glutamic acid (N-acyl-poly-L-α-glutamicacid).

Example 3A

Poly-α-L-glutamic acid-γ-(tert-butyl)ester (1 g, 5.37 mmol) wasdissolved in THF (20 mL) and catalytic DMAP (8 mg, 0.07 mol) was added.Then N—BOC-L-Phenylalanine (180 mg, 0.7 mol) was added to the mixtureand immediately after DIPC (160 μL, 1 mmol) was dropwise added. Theresulting solution was stirred for about two hours then water (2volumes) was added and a white precipitate formed immediately. Thesuspension was stirred for 30 min and filtered, then the white powderedsolid was suspended in formic acid (18 mL). The resulting mixture wasstirred for 2 h at 60° C. and formic acid was removed by distillation atreduced pressure (P=15 torr) followed by azeotropic distillation withtoluene (2×10 mL of toluene) under slight vacuum (Lakhanpal M. L.;Mandal H. G.; Lal G.; Indian J. Chem, 13, 1309 (1975)). The solidresidue was dissolved in aqueous NaHCO₃ and the resulting solution wasultrafiltered through a 5,000 Da cutoff membrane. The retentate wasconcentrated to about 30 mL and acidified with sulphuric acid until pH2-2.5. The precipitated material was stirred for 30 min and recovered byfiltration yielding, after drying under vacuum (30° C., overnight), thecorresponding N-terminus phenylalanine-capped poly-α-L-glutamic acid,i.e., N-phenylalanyl-poly-α-L-glutamic acid (530 mg, 84% recovery). The¹H-NMR was consistent with the proposed structure.

The N-terminal free amine content (clearly detectable at thepoly-α-L-glutamic acid-γ-(tert-butyl)ester stage) was no longerdetectable by ¹H-NMR analysis or by analysis following derivatizationwith o-phthalaldehyde.

M_(w)=13,300 polydispersity=1.16 as determined by GPC-MALLS analysis.

Example 3B

Poly-α-L-glutamic acid-γ-(tert-butyl)ester (1 g, 5.37 mmol) wasdissolved in THF (40 mL) and DMAP (42 mg, 0.34 mol) was added. ThenL-pyroglutamic acid (44 mg, 0.34 mol) was added to the mixture andimmediately after DIPC (106 μL, 0.7 mmol) was dropwise added. Theresulting solution was stirred for about two hours then water (2volumes) was added and a white precipitated formed immediately. Thesuspension was stirred for 30 min, then filtered and the recovered whitepowder was suspended in formic acid (18 mL). The resulting mixture wasstirred for 2 h at 60° C. and formic acid was removed by distillation atreduced pressure (P=15 torr) followed by azeotropic distillation withtoluene (2×10 mL of toluene) under slight vacuum. The solid residue wasdissolved in aqueous NaHCO₃ and the resulting solution was ultrafilteredthrough a 5,000 Da cutoff membrane. The retentate was concentrated toabout 30 mL and acidified by means of sulphuric acid until pH 2-2.5. Theprecipitated material was stirred for 30 min and recovered by filtrationyielding, after drying under vacuum (30° C., overnight), thecorresponding N-terminus pyro-L-glutamic-capped poly-α-L-glutamic acid(400 mg, 64% recovery). The ¹H-NMR was consistent with the proposedstructure and the free amine (clearly detectable at thepoly-α-L-glutamic acid-γ-(tert-butyl)ester stage) was no longerdetectable by ¹H-NMR or o-phthalaldehyde analysis.

Example 3C

Naphthalen-2-yl-acetic acid (19.7 mg, 0.106 mmol) and N-methylmorpholine (23.7 mg, 25.7 μL, 0.234 mmol) were dissolved in CH₂Cl₂ (10mL) and the mixture was cooled at 0° C.

Chlorotripyrrolidinophosphonium hexafluorophosphate (PyClop, 0.106 mmol,44.7 mg) was added to the mixture and the resulting solution was stirredfor about 1 hour, then poly-α-L-glutamic acid-γ-(tert-butyl)ester wasadded quickly (n≈100; 1 g, 0.054 mmol) and the reaction was stirred foradditional 5 hours after the removal of the cooling bath. The solventwas evaporated under reduced pressure. The crude material was dissolvedagain in THF (10 mL) and water (2 volumes) was added. The resultingsuspension was stirred for 30 min, then it was filtered and therecovered white powder was suspended in formic acid (18 mL). Theresulting mixture was stirred for 2 h at 60° C. The mixture was cooleddown and formic acid was removed by distillation at reduced pressure(P=15 torr) followed by azeotropic distillation with toluene (2×10 mLeach). The solid residue was dissolved in aqueous NaHCO₃ and theresulting solution was ultrafiltered through a 5000 Da cutoff membrane.The retentate was concentrated to about 30 mL and acidified by means ofsulfuric acid until pH 2-2.5. The precipitated material was stirred for30 min and recovered by filtration yielding, after drying under vacuum(30° C., overnight), the correspondingN-(Naphthalen-2-yl-acetyl)-poly-α-L-glutamic acid (570 mg, 82%recovery). The ¹H-NMR was consistent with the proposed structure and thefree amine (clearly detectable at the poly-α-L-glutamicacid-γ-(tert-butyl)ester stage) was no longer detectable by ¹H-NMR oro-phthalaldehyde analysis.

Example 3D

Naphthalen-2-yl-acetic acid (31.7 mg, 0.17 mmol), TEA (3.5 mg, 4.7 μL,0.034 mmol) and N,N′-disuccinimidyl carbonate (DSC, 43.6 mg 0.17 mmol),were dissolved in a 1:1 mixture of CH₂Cl₂/CH₃CN (2 mL) and the resultingmixture was stirred at room temperature for 2 hours. The mixture wasdropwise added to a poly-α-L-glutamic acid-γ-(tert-butyl)ester (n≈100; 1g, 0.054 mmol) solution in CH₂Cl₂ (10 mL) and the resulting solution wasstirred overnight. The solvent was then removed under reduced pressureand the crude material was processed in the same way as described in theExample 3C.

The recovered solid, after drying under vacuum (30° C., overnight),furnished the corresponding N-(Naphthalen-2-yl-acetyl)-poly-α-L-glutamicacid (612 mg, 88% recovery). The ¹H-NMR was consistent with the proposedstructure and the free amine (clearly detectable at thepoly-α-L-glutamic acid-γ-(tert-butyl)ester stage) was no longerdetectable by ¹H-NMR or o-phthalaldehyde analysis.

Example 3E

Poly-α-L-glutamic acid-γ-(tert-butyl)ester (n≈100; 1 g, 0.054 mmol) wasdissolved in THF (20 mL) and catalytic DMAP (8 mg, 0.07 mmol) was added.Then 2-thiophenebutyric acid (119 mg, 0.7 mmol) was added to the mixtureand immediately after DIPC (160 μL, 1 mmol) was dropwise added. Theresulting solution was stirred for about two hours then water (2volumes) was added and a white precipitate formed immediately. Thesuspension was stirred for 30 min and filtered, then the white powderedsolid was suspended in formic acid (18 mL). The resulting mixture wasstirred for 2 h at 60° C. and formic acid was removed by distillation atreduced pressure (P=15 torr) followed by azeotropic distillation withtoluene (2×10 mL of toluene) under slight vacuum. The solid residue wasdissolved in aqueous NaHCO₃ and the resulting solution was filtered on afiberglass filter (0.2 μm). The filtrate was acidified with H₂SO₄ 2Nuntil pH 2-2.5. The precipitated material was stirred for 30 min andrecovered by filtration yielding, after drying under vacuum (30° C.,overnight), the corresponding N-terminus 2-thiophenebutyroyl-cappedpoly-α-L-glutamic acid, i.e., N-(2-thiophenebutyroyl)-poly-α-L-glutamicacid (480 mg, 76% recovery). The ¹H-NMR was consistent with the proposedstructure.

The N-terminal free amine content (clearly detectable at thepoly-α-L-glutamic acid-γ-(tert-butyl)ester stage) was no longerdetectable by ¹H-NMR analysis or by analysis following derivatisationwith o-phthalaldehyde.

M_(w)=13,300, polydispersity=1.12 as determined by GPC-MALLS analysis.

According to a very similar procedure,N-(2-thiophenepropanoyl)-poly-α-L-glutamic acid andN-(2-thiophenepentanoyl)-poly-α-L-glutamic acid, were also obtained.

All of the above U.S. patents, U.S. patent application publications,U.S. patent applications, foreign patents, foreign patent applicationsand non-patent publications referred to in this specification and/orlisted in the Application Data Sheet, are incorporated herein byreference, in their entirety.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention.

1. A process for the preparation of poly-α-glutamic acid of formula (I)

wherein the symbol * indicates a chiral center and n is between 60 and310, so that the poly-α-glutamic acid has a molecular weight rangingfrom 8,000 to 40,000 Da said process comprising the steps of:polymerization of a tertiary γ-ester of α-glutamic acid N-carboxyanhydride of formula (II)

wherein the symbol * is a defined above and R is selected from t-butyl,1,1-dimethylpropyl and 1,1-dimethylbutyl in water or in an organicsolvent selected from: tetrahydrofuran, 1,4-dioxane, dimethylformamide,1,4-dioxane/DMF and 1,4-dioxane/tetrahydrofuran mixture with aninitiator selected from potassium tert-butoxide, sodium methoxide,diisopropylethylamine, 1,8-diazabicyclo[5,4,0]undec-7-ene,dimethylaminopyridine and L-glutamic acid-γ-tert-butylester, to give acompound of formula (III)

wherein * and R are as defined above and R′ is hydrogen when theinitiator is selected from diisopropylethylamine,1,8-diazabicyclo[5,4,0]undec-7-ene, 4-dimethylaminopyridine, glutamicacid dimethyl ester and glutamic acid-γ-tert-butyl ester or R′ is at-butyl or methyl group when the initiator is potassium tert-butoxideand sodium methoxide respectively; and followed by b) acid hydrolysis ofthe γ- and α-ester groups to give a compound of formula (I).
 2. Theprocess according to claim 1 wherein the solvent is 1,4-dioxane and theinitiator is 1,8-diazabicyclo[5,4,0]undec-7-ene.
 3. The processaccording to claim 1 wherein the temperature ranges from 10 to 50° C. 4.The process according to claim 2 wherein the temperature ranges from 10to 50° V.
 5. The process according to claim 1 wherein the concentrationof the tertiary γ-ester of α-glutamic acid N-carboxy anhydride rangesfrom 0.1 to 0.3 M.
 6. The process according to claim 2 wherein theconcentration of the tertiary γ-ester of α-glutamic acid N-carboxyanhydride ranges from 0.1 to 0.3 M.
 7. The process according to claim 3wherein the concentration of the tertiary γ-ester of α-glutamic acidN-carboxy anhydride ranges from 0.1 to 0.3 M.
 8. The process accordingto claim 4 wherein the concentration of the tertiary γ-ester ofα-glutamic acid N-carboxy anhydride ranges from 0.1 to 0.3 M.
 9. Theprocess according to claim 1 wherein step b) is carried out in an acidselected from trifluoroacetic acid, formic acid and water/formic acidmixtures at a temperature ranging from 20 to 60° C.
 10. The processaccording to claim 2 wherein step b) is carried out in an acid selectedfrom trifluoroacetic acid, formic acid and water/formic acid mixtures ata temperature ranging from 20 to 60° C.
 11. The process according toclaim 3 wherein step b) is carried out in an acid selected fromtrifluoroacetic acid, formic acid and water/formic acid mixtures at atemperature ranging from 20 to 60° C.
 12. The process according to claim4 wherein step b) is carried out in an acid selected fromtrifluoroacetic acid, formic acid and water/formic acid mixtures at atemperature ranging from 20 to 60° C.
 13. The process according to claim5 wherein step b) is carried out in an acid selected fromtrifluoroacetic acid, formic acid and water/formic acid mixtures at atemperature ranging from 20 to 60° C.
 14. The process according to claim6 wherein step b) is carried out in an acid selected fromtrifluoroacetic acid, formic acid and water/formic acid mixtures at atemperature ranging from 20 to 60° C.
 15. The process according to claim7 wherein step b) is carried out in an acid selected fromtrifluoroacetic acid, formic acid and water/formic acid mixtures at atemperature ranging from 20 to 60° C.
 16. The process according to claim8 wherein step b) is carried out in an acid selected fromtrifluoroacetic acid, formic acid and water/formic acid mixtures at atemperature ranging from 20 to 60° C.
 17. The process according to claim1 wherein R is t-butyl.
 18. The process according to any one of claims 1to 17 wherein the molecular weight of the poly-α-glutamic acid (I)ranges from 13,000 to 16,000 Da and the polydispersity index is ≦1.5.19. A process for the preparation of a poly-α-glutamic acid derivativeof formula (IV)

wherein the symbol * indicates a chiral center and n is an integercomprised between 60 and 310 and R₁CO— is selected from:(C₁-C₁₀)alkylcarbonyl; (C₄-C₈)cycloalkylcarbonyl;(C₂-C₆)carboxyalkylcarbonyl; (C₆-C₁₀)arylcarbonyl;(C₆-C₁₀)aryl(C₁-C₁₀)alkylcarbonyl; (C₁-C₁₀)alkyl(C₆-C₁₀)arylcarbonyl;(C₅-C₁₀)heteroarylcarbonyl and (C₅-C₁₀)heteroaryl(C₁-C₁₀)alkylcarbonylwherein the heteroaromatic ring contains one or more nitrogen, oxygen orsulphur atoms; and D- or L-amino acid and non-natural amino acidresidues; said process comprising the steps of: a) polymerization of atertiary γ-ester of an α-glutamic acid N-carboxy anhydride of formula(II)

wherein * is as defined above and R is selected from t-butyl,1,1-dimethylpropyl and 1,1-dimethylbutyl in water or in an organicsolvent selected from: tetrahydrofuran, 1,4-dioxane, dimethylformamide,1,4-dioxane/DMF and 1,4-dioxane/tetrahydrofuran mixtures with aninitiator selected from potassium tert-butoxide, sodium methoxide,diisopropylethylamine, 1,8-diazabicyclo[5,4,0]undec-7-ene,dimethylaminopyridine and L-glutamic acid-γ-tert-butylester, to give acompound of formula (III); b) reaction of a compound of formula (III)

obtained according to step a) above with a carboxylic acid R₁COOH, or anacyl chloride R¹COCl or an anhydride (R₁CO)₂O wherein R₁ is as definedabove, in the presence of a dehydrating agent to give a compound offormula (V)

and c) the hydrolysis of the compound of formula (V) to give a compoundof formula (IV).
 20. The process according to claim 19 wherein(C₁-C₁₀)alkylcarbonyl is acetyl or butyryl.
 21. The process according toclaim 19 wherein (C₄-C₈)cycloalkylcarbonyl is cyclopropylcarbonyl,cyclobutanecarbonyl, or cyclohexylcarbonyl.
 22. The process according toclaim 19 wherein (C₂-C₆)carboxyalkylcarbonyl is succinyl.
 23. Theprocess according to claim 19 wherein (C₆-C₁₀)arylcarbonyl is benzoyl,1-naphthoyl or 2-naphthoyl.
 24. The process according to claim 19wherein (C₆-C₁₀)aryl(C₁-C₁₀)alkylcarbonyl is phenylacetyl orphenylbutyryl.
 25. The process according to claim 19 wherein(C₁-C₁₀)alkyl(C₆-C₁₀)aryl carbonyl is o-, m- or p-tolyl.
 26. The processaccording to claim 19 wherein (C₅-C₁₀)heteroarylcarbonyl is nicotinoyl,N-methylpyrrole-3-carbonyl, 3-thiophenecarbonyl or 3-quinolinecarbonyl.27. The process according to claim 19 wherein(C₅-C₁₀)heteroaryl(C₁-C₁₀)alkylcarbonyl is 3-pyridylacetyl.
 28. Theprocess according to claim 19 wherein D- or L-natural amino acidresidues are those deriving from glycine, alanine, valine, leucine,isoleucine, serine, threonine, lysine, pyroglutamic acid, phenylalanine,tryptophan and cysteine.
 29. The process according to claim 28 whereinthe amino acid residue is a phenylalanine residue.
 30. The processaccording to claim 19 wherein D or L non-natural amino acid residues arethose deriving from β-alanine, α,α-dimethylglycine, α-phenylglycine,homophenylalanine, 3-amino-3-(4-methylphenyl)propionic acid, and2-(1-aminocyclopentyl)acetic acid.
 31. A poly-α-glutamic acid derivativeof formula (IV)

wherein the symbol * indicates a chiral center and n is an integercomprised between 60 and 310 and R₁CO— is selected from:(C₁-C₁₀)alkylcarbonyl; (C₄-C₈)cycloalkylcarbonyl; (C₆-C₁₀)arylcarbonyl;(C₆-C₁₀)aryl(C₁-C₁₀)alkylcarbonyl; (C₁-C₁₀)alkyl(C₆-C₁₀)arylcarbonyl;(C₅-C₁₀)heteroarylcarbonyl and (C₅-C₁₀)heteroaryl(C₁-C₁₀)alkylcarbonylwherein the heteroaromatic ring contains one or more nitrogen, oxygen orsulphur atoms; and D- or L-amino acid and non-natural amino acidresidues as well as their salts with inorganic acids or bases.
 32. Thecompound according to claim 31 wherein R₁CO is a D- or L-pyroglutamicacid residue and the content of the N-terminus free amine is lower than1% w/w, preferably lower than 0.2% w/w.
 33. A poly-α-glutamic acidderivative of formula (V)

wherein the symbol * indicates a chiral center and n is an integercomprised between 60 and 310, R is selected from t-butyl,1,1-dimethylpropyl and 1,1-dimethylbutyl, R′ is hydrogen or a t-butyl ormethyl group and R₁CO is selected from: (C₁-C₁₀)alkylcarbonyl;(C₄-C₈)cycloalkylcarbonyl; (C₆-C₁₀)arylcarbonyl;(C₆-C₁₀)aryl(C₁-C₁₀)alkylcarbonyl; (C₁-C₁₀)alkyl(C₆-C₁₀)arylcarbonyl;(C₅-C₁₀)heteroarylcarbonyl and (C₅-C₁₀)heteroaryl(C₁-C₁₀)alkylcarbonylwherein the heteroaromatic ring contains one or more nitrogen, oxygen orsulphur atoms; and D- or L-amino acid and non-natural amino acidresidues.
 34. The poly-α-glutamic acid derivative according to claim 31or claim 33 substituted at the —NH₂ terminus with a group which is ableto be further functionalized with a moiety suitable for modulating thepharmacokinetic properties of poly-α-glutamic acid.